The method of continuous variation, also known as the Job plot, has long been used for determining the stoichiometry of two interacting components. A general review of the method with theoretical extension to cooperative binding systems has been presented by Huang (Huang, C.Y., Methods Enzymol. 87, 509-512, 1982). From the equations derived by Huang, it became apparent that anomalous binding ratios, usually higher than the true one, may be observed for cooperative systems under certain conditions. This feature, however, was not highlighted in the review at the time. With the Job plot, the total molar concentration of two interacting components is held constant, while their molar ratios are continuously varied. A measurable parameter that is linearly proportional to the complex(es) formed is plotted against the mole fractions of the reactants to generate a curve. The binding stoichiometry is then determined from the ratio of the mole fraction of the two components found at either the maximum of the curve, Rm, or the intersection of the two limiting slopes of the curves as they approach zero, Ri. In carrying out the experiments, the concentration of one of the reactants can be calculated in terms of its binding equivalents in addition to its molar concentration. In this project, it was shown that distinctive patterns of Rm and Ri as a function of total reactant concentration calculated in terms of moles or binding equivalents could be assigned to different binding modes--namely, the equivalent and noninteracting binding sites; the one step or infinite cooperativity; and the step-wise cooperative binding. Thus, these patterns permit differentiation of various interaction mechanisms. The binding of Fe(II) with ferrozine was tested using the theory and experimental design generated from this project. The system was chosen because of the role of iron in oxidation in the cell. Anomalous binding ratio was observed, as predicted, in one of the experiments. The binding mechanism was found most consistent with infinite cooperativity with one Fe(II) chelating three ferrozines with a dissociation constant of 8 micromolar.